Phase II is the development of a multiple-array oligonucleotide synthesis instrument that will greatly decrease the cost and simplify construction of large double-stranded DNA segments. This novel technology uses less than 1/10th of the reagents of conventional DNA synthesizers, and produces 100-200 nanograms of finished oligo per reaction. The process enables the synthesis of up to 10,000 overlapping oligos that can be assembled into double-stranded DNA by annealing and ligation. The methodology combines conventional oligo nucleotide synthesis chemistry with novel lithographic valving technology to deliver synthesis reagents to a large array of individual reaction chambers. In Phase I, a prototype of this system was successfully tested. This device uses a series of computer generated valving mats that effect opening or closing of miniaturized oligo synthesis reaction chambers. The prototype allowed synthesis of (800) 34-base oligos at a reagent cost of ~$0.012 per base. These overlapping oligos were annealed and ligated to form a predicted 13,600 bp fragment of dsDNA coding for active antibiotic resistance genes. The error rate in the resulting synthetic dsDNA was less than one in 1300 bases. In Phase II, the large array oligo synthesis method will be further developed and validated using a 10,000-reaction well prototype. Phase II work includes testing of various thin- film materials for the valving mats, construction and testin of a prototype for simultaneous synthesis of (10,000) overlapping 34-mer oligos in an 80 x 125 array. The resulting oligos will be annealed and ligated to form a set of gene fragments that will be cloned into a plasmid vectors, sequenced, and assayed for activity to validate the overall process. Additional Phase II trials will be carried out to automate the litho mat changing and registration process, as well as reduce the amount of reagents consumed in the synthesis. Synthesized cloned fragments from the above will be assembled into 100 kb or greater dsDNAs using the Gibson assembly. The technology is predicted to generate large-arrays of oligos and dsDNA at a cost of less than $0.01 per base pair. This cost will enable our company offer custom oligo production and gene synthesis at 1/10 that current suppliers' prices. The products that result from this work address the rapidly growing field of genome and metabolic pathway manipulation that is important to drug discovery, as well as the development of industrially useful bacteria. The technology is projected to generate $40 million in annual sales. MacConnell Research can directly market products and services developed by this work after Phase II.